Gaussia luciferase is a secretory enzyme produced from the deep-sea copepoda, Gaussia princeps, and the luminescence system catalyzed by Gaussia luciferase is simple and the luminescence reaction is performed only under the presence of oxygen and a substrate (a luciferin such as a coelenterazine, etc.).
By comparison with the luminescence reaction catalyzed by firefly luciferase, the luminescence reaction catalyzed by Gaussia luciferase is simple and emits strong blue light. On the other hand, firefly luciferase requires ATP and magnesium ions for the luminescence reaction. From this reason, Gaussia luciferase expects to be used in various applications in the future.
Gaussia luciferase is a simple protein having the signal peptide sequence of 17 amino acid residues at the amino terminus for secretion and the catalytic domain consisting of 168 amino acid residues. Gaussia luciferase is a unique luciferase having 10 cysteine residues in a molecule, in which the content of cysteines is approximately 6% in the total amino acid. It has been reported that an intramolecular —S—S— bond is critical to retain the catalytic ability for the luminescence activity of Gaussia luciferase. By treatment of Gaussia luciferase with reducing agents such as mercaptoethanol, dithiothreitol, etc., the luminescence activity of Gaussia luciferase was lost completely (Inouye, S. & Sahara, Y. (2008) Biochem. Biophys. Res. Commun. 365, 96-101). However, there is little information on the positions of the intramolecular —S—S-bond or free thiol groups in Gaussia luciferase.
By the chemical modification of Gaussia luciferase with a ligand using the intramolecular thiol groups of Gaussia luciferase, namely, by introducing a ligand into Gaussia luciferase via thiol groups of amino acid residue in Gaussia luciferase, the ligand-conjugated Gaussia luciferase can be obtained. Using the ligand-conjugated Gaussia luciferase and relying on the luminescence reaction of Gaussia luciferase, it is considered to detect a substance capable of specifically binding to the ligand. However, there is no report so far on such a case that Gaussia luciferase was directly conjugated with the ligand by chemical modification.
The present inventors found that when a ligand is introduced into Gaussia luciferase via the thiol groups from the amino acids which constitute Gaussia luciferase, the catalytic ability of the ligand-conjugated Gaussia luciferase for the luminescence activity is markedly reduced. The inventors also attempted to introduce a ligand into Gaussia luciferase via amino groups or carboxyl groups, other than the thiol groups, derived from the amino acids which constitute Gaussia luciferase. However, the catalytic ability of Gaussia luciferase for the luminescence activity was likewise markedly reduced. As such, it was impossible to obtain the ligand-conjugated Gaussia luciferase by chemical modification while maintaining the original luminescence intensity of Gaussia luciferase. This is considered to be because the protein catalytic domains associated with the luminescence reaction of Gaussia luciferase would be affected by introducing the ligand via the thiol, amino or carboxyl groups derived from the amino acids in the molecule of Gaussia luciferase, and the luminescence reaction would be inhibited.
On the other hand, it is considered that when cysteine residues are introduced into the molecule or at the amino or carboxyl terminus of Gaussia luciferase, the formation of correct intramolecular —S—S— bonds by refolding of a protein expressed in cells would be hindered.
The only ligand-conjugated Gaussia luciferase reported is biotinylated Gaussia luciferase modified with a biotinylation enzyme. This luciferase was prepared by expressing the fused Gaussia luciferase gene having a modifiable biotin recognition sequence in Escherichia coli followed by biotinylation with an enzyme present in E. coli (Verhaegen, M. & Christopoulos, T. K. (2002) Anal. Chem. 74, 4378-4385). According to an enzymatic modification in living cells as in this method, however, it is difficult to supply biotinylated Gaussia luciferase uniformly in large amounts. Furthermore, according to this method the ligand which can be introduced into Gaussia luciferase is biotin alone, but other ligands such as avidin, streptavidin, enzymes, antibodies, antigens, nucleic acids, polysaccharides, receptors or the like or fluorescent substances, etc. cannot be introduced into Gaussia luciferase. The ligand which can be introduced is biotin alone and its application is limited.